It is now a routine procedure to generate transgenic plants, either through Agrobacterium-mediated transformation or direct DNA transfer methods (Gelvin, Curr. Opin. Biotechnol., 9:227-232 (1998); Komari et at., Curr. Opin. Plant Biol., 1:161-165 (1998); Tyagi et al., Crit. Rev. Biotechnol., 19:41-79 (1999)). Many commercially important dicot and monocot species have been transformed. However, while the transformation procedure itself is no longer considered a limiting step, the recovery of useful transgenic lines is hampered by variable transgene expression levels and transgene silencing (Flavell, Proc. Natl. Acad. Sci. USA, 91:3490-3496 (1994); Finnegan, Ann. Rev. Plant Physiology, 49:223-247 (1998)). Complete or partial transgene silencing has been attributed to a number of factors including the copy number of the integrated genes (Matzke and Matzke, Plant Physiol., 107:679-685 (1995) and Matzke et al. Mol. Gen. Genet., 244:219-229 (1994)), the position of transgene integration (Hobbs et al., Plant Mol. Biol., 15:851-864 (1990); Peach and Velten, Plant Mol. Biol., 17:49-60 (1991); Matzke and Matzke, Curr. Opin. Plant Biol., 1:142-148 (1998)), the configuration and structure of the transgenic locus (Stam et al., Plant J., 12:63-82 (1997)), the structural integrity of individual transgenes (Kohli et al., 1998) and the genetic properties of the host plant, (e.g., genetic background, ploidy and zygosity) (Beaujean et al., Mol. Gen. Genet., 260:362-371 (1998)).
Whole plasmid transformation is still almost universal in plant systems. This may trace its roots to the requirement for Agrobacterium vector sequences in Agrobacterium-mediated transformation. The Agrobacterium vector carries essential vir genes required for T-DNA excision, transfer and integration. In previously described transformation methods, the transgene cassette encoding a desired product is cloned into a vector, e.g., a plasmid, a virus or an Agrobacterium Ti plasmid, and the entire vector containing the transgene cassette is introduced into the plant cell. However, vector sequences serve no required purpose for DNA transfer and integration in direct DNA transfer procedures such as, e.g., particle bombardment. The methods of this invention generate transgenic plants that display stable transgene expression at a higher frequency than produced by previously described methods. In embodiments wherein two or more transgenes are simultaneously introduced into the plants, the frequency of transgene co-expression is higher than the frequency of co-expression produced by previously described methods. The transgenic plants produced by the methods described herein exhibit simple integration patterns, low copy number of the transgene and in general do not exhibit transgene silencing.